PROJECT SUMMARY This proposal aims to develop a novel and practical therapeutic platform to revascularize ischemic tissues. In peripheral artery disease (PAD), stenting and bypass fail to permanently relieve symptoms, and in 30% of cases, amputation must be performed. These patients exhibit mortality rates of 25% within a year of surgery and 60% within five years. Cases of critical limb ischemia (CLI), the most severe form of PAD, cost over $50,000 per patient per year, with direct billing of $25 billion per year in the U.S. Our lab has developed a novel approach in which a massive number of injectable microtissues (iMTs) with blood-vessel building blocks can be produced and gently harvested. In vitro, we demonstrated over a quarter billion cells can be easily coaxed into spheroids, and in vivo, we injected therapeutic quantities into wildtype and diseased mouse models. Our approach uses dissolvable alginate as a sacrificial material for microwells to generate microtissue reproducibly in a manner suited for mass production. In preliminary work, we have validated this approach by observing the self-organization of thousands of microtissues in vitro and in vivo, including rapid integration with the host's vascular network and re-perfusion. Instead of weeks, our results so far have shown that host re- vascularization and re-perfusion occurs within days. This R01 grant will allow us to automate the production of the microtissues, study the extent to which we can control the architecture of the ensuing in vivo vascular network, and most significantly, fully explore the therapeutic potential of this novel platform via a large, properly-powered number of treatment and control groups.